EP4328232A1 - Electrochemical biosensor, or sensing membrane for electrochemical biosensor containing transition metal complex or oxidation-reduction polymer - Google Patents

Electrochemical biosensor, or sensing membrane for electrochemical biosensor containing transition metal complex or oxidation-reduction polymer Download PDF

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EP4328232A1
EP4328232A1 EP22792013.9A EP22792013A EP4328232A1 EP 4328232 A1 EP4328232 A1 EP 4328232A1 EP 22792013 A EP22792013 A EP 22792013A EP 4328232 A1 EP4328232 A1 EP 4328232A1
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oxidation
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Hyunseo Shin
Bona YANG
Geunhee KANG
Su-Jin Kim
Areum YU
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i Sens Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/002—Osmium compounds
    • C07F15/0026—Osmium compounds without a metal-carbon linkage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1468—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means
    • A61B5/1473—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means invasive, e.g. introduced into the body by a catheter
    • C—CHEMISTRY; METALLURGY
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    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0046—Ruthenium compounds
    • C07F15/0053—Ruthenium compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/02—Iron compounds
    • C07F15/025—Iron compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F226/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen
    • C08F226/06—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen by a heterocyclic ring containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
    • C08F230/04—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing a metal
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/02—Alkylation
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/42—Introducing metal atoms or metal-containing groups
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/004—Enzyme electrodes mediator-assisted
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Definitions

  • the present invention relates to a transition metal complex having a bidentate ligand comprising pyrazole, triazole, tetrazole, oxadiazole or thiadiazole, or the like and an electrochemical biosensor using the same.
  • biosensors using enzymes are chemical sensors used to selectively detect and measure chemical substances contained in a sample by using a functional substance of a living organism or a biological detection function in which a living organism such as microorganisms, and the like sensitively reacts with specific substances, and mostly, have been developed for a medical measurement use, and in addition, research is also being actively conducted in applications in the field of food engineering and environmental measurement.
  • Periodic measurement of blood glucose is very important in management of diabetes, and therefore, various blood glucose meters are being manufactured so that blood glucose can be easily measured using a portable measuring instrument.
  • the principle of operation of such biosensors is based on an optical method or electrochemical method, and such electrochemical biosensors can reduce an effect by oxygen differently from biosensors by the conventional optical method, and have an advantage of being able to be used without separate retreatment of a sample, even though the sample is turbid. Therefore, various kinds of electrochemical biosensors having accuracy and precision are widely used.
  • electrochemical blood glucose sensors mainly use enzyme electrodes, and more specifically, have a structure in which glucose oxidase is fixed by a chemical or physical method on an electrode that can convert electrical signals.
  • Such electrochemical blood glucose sensors are based on the principle of measuring a glucose concentration in an analyte by measuring the current generated by transferring electrons generated when glucose in the analyte such as blood and the like is oxidized by an enzyme.
  • biosensors using enzyme electrodes as the distance from the active center of the enzyme is too far, a problem that it is not easy to directly transfer electrons generated when a substrate is oxidized into an electrode is caused.
  • an oxidation-reduction mediator that is, an electron transfer mediator is necessarily required.
  • an electron transfer mediator that is, an electron transfer mediator is necessarily required.
  • the most commonly used electron transfer mediator includes potassium ferricyanide [K 3 Fe(CN) 6 ], and due to inexpensive price and great reactivity, it is useful for all sensors using FAD-GOX, PQQ-GDH or FAD-GDH.
  • sensors using this electron transfer mediator have measurement errors due to interfering substances such as uric acid or gentisic acid present in blood, and are easy to be deteriorated by the temperature and humidity, so particular attention should be taken in manufacturing and storage, and there are difficulties of accurately detecting glucose at a low concentration due to changes in background current after long-term storage.
  • Hexamine ruthenium chloride [Ru(NH 3 ) 6 Cl 3 ] has higher oxidation-reduction stability than ferricyanide, and therefore, biosensors using this electron transfer mediator have advantages of easy manufacturing and storage, and high stability due to small background current changes even during long-term storage, but there are disadvantages that it is difficult to manufacture it as a commercially useful sensor, since the reactivity does not match to use with FAD-GDH.
  • a continuous glucose monitoring (CGM) system is used to manage disease such as diabetes by continuously observing blood glucose, but in conventional enzyme sensors, collecting blood from a fingertip causes significant pain due to a needle during blood-gathering, so the measurement frequency is limited, and thus it cannot be used for such CGM.
  • CGM continuous glucose monitoring
  • an oxidation-reduction polymer of an enzyme sensor is prepared and used mainly by fixing a transition metal electron transfer mediator comprising bipyridine and bisimidazole ligands on the polymer skeleton.
  • a transition metal complex having a bidentate ligand comprising a heterocyclic compound including pyrazole, triazole, tetrazole, oxadiazole or thiadiazole, or the like in addition to bipyridine and bisimidazole ligands, thereby completing the present invention.
  • the present invention provides a transition metal complex or a salt compound thereof, which has a bidentate ligand comprising a heterocyclic structure such as pyrazole, triazole, tetrazole, oxadiazole or thiadiazole, or the like, in which synthesis of various derivatives and introduction of a functional group are easy.
  • a bidentate ligand comprising a heterocyclic structure such as pyrazole, triazole, tetrazole, oxadiazole or thiadiazole, or the like, in which synthesis of various derivatives and introduction of a functional group are easy.
  • the transition metal complex may be a transition metal complex or a salt compound thereof having a bidentate ligand comprising pyridine; and one structure selected from the group consisting of pyrazole, triazole, tetrazole, oxadiazole and thiadiazole.
  • Another object of the present invention is to provide an oxidation-reduction polymer, comprising the transition metal complex or salt compound thereof.
  • Another object of the present invention is to provide the transition metal complex or salt compound thereof and/or an oxidation-reduction polymer comprising the same to a device.
  • the device may be a device for an electron transfer mediator, specifically, an electrochemical biosensor.
  • the device may be insertable into the body.
  • the electrochemical biosensor may be a blood glucose sensor.
  • sensing membrane for an electrochemical biosensor comprising an enzyme capable of oxidizing-reducing a liquid biological sample; and the transition metal complex or salt compound thereof and/or an oxidation-reduction polymer comprising the same.
  • the present invention provides a transition metal complex or a salt compound thereof, having a bidentate (hereinafter, also called “bidentate”) comprising pyridine; and one structure selected from the group consisting of pyrazole, triazole, tetrazole, oxadiazole and thiadiazole.
  • the transition metal complex may be a compound of the following Chemical formula 1.
  • Chemical formula 1 [M(L) a (X 1 ) b ] c d(X 2 ) in the formula,
  • halo or halogen means for example, fluoro, chloro, bromo and iodo.
  • alkyl means an aliphatic hydrocarbon radical, and includes all linear or branched hydrocarbon radicals.
  • the aliphatic hydrocarbon having 1 to 6 carbon atoms includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl, but not limited thereto.
  • the alkyl may mean alkyl having 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, 1 to 2 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms, 3 to 6 carbon atoms, 3 to 5 carbon atoms, 3 to 4 carbon atoms, 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms.
  • alkoxy represents a -O-alkyl or alkyl-O- group, and herein, the alkyl group is the same as defined above. For example, those such as methoxy, ethoxy, n-propoxy, n-butoxy, and t-butoxy are included, but not limited thereto.
  • the alkoxy group may be substituted or unsubstituted with at least one appropriate group.
  • amino represents -NH 2
  • nitro represents - NO 2
  • aryl refers to a monovalent aromatic ring having for example, 6 to 20 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms, induced by removing one hydrogen atom in one carbon atom in a parent aromatic ring system.
  • the aryl may comprise a bicyclic radical comprising an aromatic ring fused with a saturated or partially unsaturated ring.
  • An exemplary aryl group may include radicals induced from benzenes (phenyl), substituted phenyl, biphenyl, naphthyl, tetrahydronaphthyl, fluorenyl, toluyl, naphthalenyl, anthracenyl, indenyl, indanyl, and the like, but not limited thereto.
  • the aryl group may be substituted or unsubstituted with at least one appropriate group.
  • substitution may be, unless particularly mentioned, that at least one hydrogen atom is one kind to three kinds selected from the group consisting of halogen atoms (for example, F, Cl, Br, or I), cyano group, hydroxyl group, thiol group, nitro group, amino group, imino group, azido group, amidino group, hydrazine group, hydrazono group, oxo group, carbonyl group, carbamyl group, ester group, ether group, carboxyl group, or salts thereof, sulfonic acid or salts thereof, phosphoric acid or salts thereof, alkyl group having 1 to 6 carbon atoms, haloalkyl group having 1 to 6 carbon atoms, alkenyl group having 2 to 6 carbon atoms, haloalkenyl group having 2 to 6 carbon atoms, alkylnyl group having 2 to 6 carbon atoms, haloalkylnyl group having 2 to 6 carbon carbon carbon carbon carbon carbon carbon carbon
  • the transition metal complex provided in the present description may be a compound of the following Chemical formula 1.
  • [Chemical formula 1] [M(L) a (X 1 ) b ] c d(X 2 ) in the formula,
  • the pyridine may be unsubstituted, or substituted with at least one kind (for example, 1 to 4, 1, 2, 3, or 4) selected from the group consisting of C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, and C 1-4 alkylamino group.
  • the pyrazole, triazole, tetrazole, oxadiazole, or thiadiazole may be each unsubstituted, or substituted with at least one kind (for example, 1 to 3, 1, 2, or 3) selected from the group consisting of C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1 - 4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, and C 1-4 alkylamino group.
  • the R' 4 may be hydrogen or substituted or unsubstituted C 1-4 alkyl, and the n' may be an integer selected from 1 to 4, for example, 1, 2, 3, or 4.
  • the C 1-4 alkyl group, alkoxy group, or alkylamino group may mean an alkyl group, alkoxy group, or alkylamino group having 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 4, 3 to 4, 1, 2, 3, or 4 carbon atoms.
  • the C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, or C 1-4 alkylamino group may be unsubstituted, or substituted.
  • a hydrogen atom may be substituted with a halogen atom of F, Cl, Br or I, cyano group, hydroxy group, thiol group, nitro group, amino group, imino group, azido group, amidino group, hydrazine group, hydrazono group, oxo group, carbonyl group, carbamyl group, ester group, ether group, carboxyl group or salt thereof, sulfonic acid group or salt thereof, or phosphoric acid or salt thereof.
  • the transition metal complex may be a compound of the following Chemical formula 2. in the formula,
  • the C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, or C 1-4 alkylamino group may be unsubstituted, or substituted, respectively, and the substituted case is as described above.
  • the transition metal complex may be a compound selected from the following Chemical formula 3 to Chemical formula 25.
  • the transition metal complex according to the present invention may comprise a transition metal complex in an oxidized state, specifically, a trivalent osmium complex or a divalent osmium complex.
  • a transition metal complex used for oxidation treatment a commonly used oxidizing agent may be used, and the example of the oxidizing agent may be at least one selected from the group consisting of NaOCI, H 2 O 2 , O 2 , Os, PbO 2 , MnO 2 , KMnO 4 , ClO 2 , F 2 , Cl 2 , H 2 CrO 4 , N 2 O, Ag 2 O, OsO 4 , H 2 S 2 O 8 , Ceric ammonium nitrate (CAN), pyridinium chlorochromate, and 2,2'-Dipyridyldisulfide.
  • the transition metal complex comprises compounds in an oxidized state and a reduced state
  • a transition metal complex in an oxidized state or a salt compound thereof may
  • the transition metal complex according to the present invention may be in a form of an appropriate counter ion and/or a salt compound having an ion, and the salt compound may have high solubility in water, aqueous solution or an organic solvent.
  • the salt compound when it consists of small counter anions such as F - , Cl - and Br, and the like, it tends to be highly soluble in water or aqueous solution, and when it consists of large counter anions such as hexafluorophosphate (PF 6 - ) and tetrafluoroborate (BF 4 - ) and the like, it tends to be highly soluble in an organic solvent.
  • the example of the counter anion may be at least one selected from halide selected from the group consisting of F, Cl, Br and I, hexafluorophosphate and tetrafluoroborate.
  • the present invention provides an oxidation-reduction polymer, comprising the transition metal complex or salt compound thereof, and comprising a polymer skeleton such as polyvinyl imidazole (PVI) and polyvinyl pyridine (PVP) and the like.
  • a polymer skeleton such as polyvinyl imidazole (PVI) and polyvinyl pyridine (PVP) and the like.
  • the oxidation-reduction polymer may be a compound of the following Chemical formula 26 or Chemical formula 27: In the formula,
  • the pyridine may be unsubstituted, or substituted with at least one (for example, 1 kind, 2 kinds, 3 kinds, or 4 kinds) selected from the group consisting of C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, and C 1-4 alkylamino group.
  • the pyrazole, triazole, tetrazole, oxadiazole, or thiadiazole may be unsubstituted, or substituted with at least one (for example, 1 kind, 2 kinds, or 3 kinds) selected from the group consisting of C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, and C 1-4 alkylamino group.
  • the oxidation-reduction polymer may be a compound selected from the following Chemical formula 28 to Chemical formula 45: and in the formula, m or o is the same as defined in Chemical formula 26 or Chemical formula 27 above.
  • the oxidation-reduction polymer further comprises a crosslinkable functional group, and may be a compound of the following Chemical formula 46 or Chemical formula 47.
  • a crosslinkable functional group may be a compound of the following Chemical formula 46 or Chemical formula 47.
  • a D is one kind selected from the group consisting of primary and secondary amine groups, ammonium group, halogen group, epoxy group, azide group, acrylate group, alkenyl group, alkynyl group, thiol group, isocyanate, alcohol group, silane group, and and
  • the pyridine may be unsubstituted, or substituted with at least one (for example, 1 kind, 2 kinds, 3 kinds, or 4 kinds) selected from the group consisting of C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, and C 1-4 alkylamino group.
  • the pyrazole, triazole, tetrazole, oxadiazole, or thiadiazole may be unsubstituted, or substituted with at least one (for example, 1 kind, 2 kinds, or 3 kinds) selected from the group consisting of C 1-4 alkyl group, C 1-4 alkoxy group, -(CH2)-O-C 1-4 alkyl group, -(CH2CH2)-O-C 1-4 alkyl group, and C 1-4 alkylamino group, respectively.
  • the oxidation-reduction polymer may be a compound selected from the following Chemical formula 48 to Chemical formula 60.
  • a device comprising the transition metal complex or salt compound thereof; or the oxidation-reduction polymer.
  • the device may be an electrochemical biosensor.
  • the device may be insertable in the body, and specifically, may be an electrochemical biosensor insertable in the body.
  • the electrochemical biosensor may be a blood glucose sensor, for example, an electrochemical glucose (blood glucose) sensor.
  • the electrochemical biosensor may be a continuous blood glucose monitoring sensor.
  • the present invention may comprise for example, an electrode, an insulator, a substrate, a sensing layer comprising the oxidation-reduction polymer and oxidoreductase, a diffusion layer, a protection layer, and the like.
  • an electrode 2 kinds of electrodes such as a working electrode and a counter electrode may be comprised, and 3 kinds of electrodes such as a working electrode, a counter electrode and a reference electrode may be comprised.
  • the biosensor according to the present invention may be an electrochemical biosensor manufactured by drying after applying a reagent composition comprising the transition metal complex or salt compound thereof; or the oxidation-reduction polymer, and an enzyme capable of oxidizing-reducing a liquid biological sample, on a substrate having at least two, preferably, two or three electrodes.
  • a planar electrochemical biosensor characterized in that a working electrode and a counter electrode are equipped on the opposite side to each other of a substrate, and a sensing membrane comprising the transition metal complex or oxidation-reduction polymer of the present invention is laminated on the working electrode, and an insulator, a diffusion layer and a protection layer are laminated in order on both sides of the substrate in which the working electrode and counter electrode are equipped, in the electrochemical biosensor.
  • the substrate may be made of at least one material selected from the group consisting of PET (polyethylene terephthalate), PC (polycarbonate) and PI (polyimide).
  • a carbon, gold, platinum, silver or silver/silver chloride electrode may be used as the working electrode.
  • the counter electrode plays a role of the reference electrode, so as the counter electrode, a gold, platinum, silver or silver/silver chloride electrode may be used, and in case of the electrochemical biosensor with 3 electrodes including even the reference electrode, as the reference electrode, a gold, platinum, silver or silver/silver chloride electrode may be used, and as the counter electrode, a carbon electrode may be used.
  • Nafion, cellulose acetate and silicone rubber may be used, and as the protection layer, silicone rubber, polyurethane, polyurethane-based copolymer, and the like may be used, but not limited thereto.
  • the type of the enzyme comprised in the reagent composition of the present invention it may be applied for a biosensor for quantification of various materials such as cholesterol, lactate, creatinine, hydrogen peroxide, alcohol, amino acid, and glutamate.
  • sensing membrane for an electrochemical biosensor comprising an enzyme capable of oxidizing-reducing a liquid biological sample; and the transition metal complex or salt compound thereof; or the oxidation-reduction polymer as an electron transfer mediator.
  • the liquid biological sample may be one or more, two or more, three or more, four or more, or five or more selected from the group consisting of for example, a patient's tissue fluid, blood, cells, plasma, serum, urine, cyst liquid and saliva, but not limited thereto.
  • the enzyme may comprise at least one oxidoreductase selected from the group consisting of dehydrogenase, oxidase, and esterase; or at least one oxidoreductase selected from the group consisting of dehydrogenase, oxidase, and esterase and at least one cofactor selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), and pyrroloquinoline quinone (PQQ).
  • FAD flavin adenine dinucleotide
  • NAD nicotinamide adenine dinucleotide
  • PQQ pyrroloquinoline quinone
  • Oxidoreductase collectively refers to enzymes catalyzing oxidation-reduction reactions of a living body, and in the present invention, it means an enzyme reduced by reacting with a target substance, for example, in case of the biosensor, a target substance to be measured.
  • the enzyme reduced as such reacts with an electron transfer mediator, and the target substance is quantified by measuring the change in current generated then, and the like.
  • the oxidoreductase usable in the present invention may be at least one selected from the group consisting of various kinds of dehydrogenase, oxidase, esterase, and the like, and depending on the target substance for oxidation-reduction or detection, among enzymes belonging to the above enzyme group, an enzyme using the target substance as a substrate may be selected and used.
  • the oxidoreductase may be at least one selected from the group consisting of glucose dehydrogenase, glutamate dehydrogenase, glucose oxidase, cholesterol oxidase, cholesterol esterase, lactate oxidase, ascorbic acid oxidase, alcohol oxidase, alcohol dehydrogenase, bilirubin oxidase and the like.
  • the oxidoreductase may comprise a cofactor playing a role of storing hydrogen stolen by oxidoreductase from a target substance to be measured (for example, target substance), and for example, it may be at least one selected from the group consisting of flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), pyrroloquinoline quinone (PQQ) and the like.
  • FAD flavin adenine dinucleotide
  • NAD nicotinamide adenine dinucleotide
  • PQQ pyrroloquinoline quinone
  • glucose dehydrogenase may be used as the oxidoreductase
  • the glucose dehydrogenase may be flavin adenine dinucleotide- glucose dehydrogenase (FAD-GDH) comprising FAD as the cofactor, and/or nicotinamide adenine dinucleotideglucose dehydrogenase comprising FAD-GDH as the cofactor.
  • FAD-GDH flavin adenine dinucleotide- glucose dehydrogenase
  • FAD-GDH flavin adenine dinucleotide- glucose dehydrogenase
  • nicotinamide adenine dinucleotideglucose dehydrogenase comprising FAD-GDH as the cofactor
  • the usable oxidoreductase may be at least one selected from the group consisting of FAD-GDH (for example, EC 1.1.99.10, etc.), NAD-GDH (for example, EC 1.1.1.47, etc.), PQQ-GDH (for example, EC1.1.5.2, etc.), glutamate dehydrogenase (for example, EC 1.4.1.2, etc.), glucose oxidase (for example, EC 1.1.3.4, etc.), cholesterol oxidase (for example, EC 1.1.3.6, etc.), cholesterol esterification enzyme (for example, EC 3.1.1.13, etc.), lactate oxidase (for example, EC 1.1.3.2, etc.), ascorbic acid oxidase (for example, EC 1.10.3.3, etc.), alcohol oxidase (for example, EC 1.1.3.13, etc.), alcohol dehydrogenase (for example, EC 1.1.1.1, etc.), bil
  • the oxidoreductase is glucose dehydrogenase which can maintain activity of 70% or more in a 37°C buffer solution for 1 week.
  • the sensing membrane according to the present invention may contain 20 to 700 parts by weight, for example, 60 to 700 parts by weight, or 30 to 340 parts by weight of the oxidation-reduction polymer based on 100 parts by weight of oxidoreductase.
  • the content of the oxidation-reduction polymer may be appropriately adjusted depending on the activity of the oxidoreductase.
  • the sensing membrane according to the present invention may further comprise a carbon nanotube for an increase of membrane performance.
  • the carbon nanotube may further increase performance of the sensing membranes, as the electron transfer speed increases, when used with a transition metal complex, particularly, osmium.
  • the sensing membrane according to the present invention may further comprise a crosslinking agent.
  • the sensing membrane according to the present invention may additionally comprise at least one additive selected from the group consisting of surfactants, water-soluble polymers, quaternary ammonium salts, fatty acids, thickeners and the like, for a role of a dispersant during dissolving a reagent, an adhesive during preparing a reagent, a stabilizer during long-term storage, and the like.
  • the surfactant may play a role of making a composition is evenly spread and aliquoted in a uniform thickness on an electrode, when the composition is aliquoted.
  • the surfactant at least one selected from the group consisting of Triton X-100, sodium dodecyl sulfate, perfluorooctane sulfonate, sodium stearate, and the like may be used.
  • the reagent composition according to the present invention may contain the surfactant in an amount of 3 to 25 parts by weight, for example, 10 to 25 parts by weight, based on 100 parts by weight of oxidoreductase, in order to allow it to appropriately perform a role that the reagent is evenly spread on the electrode when the reagent is aliquoted, and the reagent is aliquoted in a uniform thickness.
  • the surfactant when oxidoreductase with activity of 700 U/mg is used, it may contain 10 to 25 parts by weight of the surfactant based on 100 parts by weight of oxidoreductase, and when the activity of oxidoreductase becomes higher than this, the content of the surfactant may be adjusted lower than this.
  • the water-soluble polymer is a polymer support of the reagent composition and may perform a role of helping stabilization and dispersing of an enzyme.
  • the water-soluble polymer at least one selected from the group consisting of polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyperfluoro sulfonate, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), carboxy methyl cellulose (CMC), cellulose acetate, polyamide, and the like may be used.
  • the reagent composition according to the present invention may contain the water-soluble polymer in an amount of 10 to 70 parts by weight, for example, 30 to 70 parts by weight, based on 100 parts by weight of oxidoreductase, in order to sufficiently and appropriately exhibit the role of helping stabilization and dispersing of oxidoreductase.
  • the water-soluble polymer in an amount of 10 to 70 parts by weight, for example, 30 to 70 parts by weight, based on 100 parts by weight of oxidoreductase, in order to sufficiently and appropriately exhibit the role of helping stabilization and dispersing of oxidoreductase.
  • oxidoreductase with activity of 700U/mg when oxidoreductase with activity of 700U/mg is used, it may contain 30 to 70 parts by weight of the water-soluble polymer based on 100 parts by weight of oxidoreductase, and when the activity of the oxidoreductase is higher than this, the content of the water-soluble poly
  • the water-soluble polymer may have a weight average molecular weight of about 2,500g/mol to 3,000,000g/mol, for example, about 5,000g/mol to 1,000,000g/mol, in order to effectively perform the role of helping stabilization and dispersing of the support and enzyme.
  • the thickener plays a role of firmly attaching a reagent to an electrode.
  • the thickener at least one selected from the group consisting of Natrosol, diethylaminoethyl-dextran hydrochloride (DEAE-Dextran hydrochloride) and the like may be used.
  • the electrochemical sensor according to the present invention may contain the thickener in an amount of 10 to 90 parts by weight, for example, 30 to 90 parts by weight, based on 100 parts by weight of oxidoreductase.
  • oxidoreductase with activity of 700U/mg when used, it may contain 30 to 90 parts by weight of the thickener based on 100 parts by weight of oxidoreductase, and when the activity of the oxidoreductase is higher than this, the content of the thickener may be adjusted lower than this.
  • the transition metal complex and oxidation-reduction polymer according to the present invention can easily adjust a potential value depending on the type of ligand introduced, and the size of the ligand is minimized than the conventional bipyridinebased one, so the electron transfer speed is increased, and therefore, the electrochemical biosensor in which this is applied has an advantage of rapid and economical detection.
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and pyrazole 4.7 g (69 mmol) and potassium butoxide 9.3 g (83 mmol) were added, and dissolved in anhydrous dimethylsulfoxide 40mL in an argon gas atmosphere.
  • 2-fluoropyrifine 8.0 g (83 mmol) was added and heated to 100 °C in an argon gas atmosphere and stirred for 4 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3). Organic layers were collected and dried with magnesium sulfate and concentrated under reduced pressure to obtain a transparent, colorless solid. (7.2 g, 72%)
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and pyrazole 2.0 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere. 2-fluoro-5-methyl pyridine 5.0 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 4 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and pyrazole 2.0 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere.
  • 2-bromo-4-methoxy pyridine 6.7 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 8 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and pyrazole 2.0 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere. 2-bromo-4-methyl pyridine 6.2 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 8 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 3-methylpyrazole 2.5 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere.
  • 2-bromo-4-methyl pyridine 6.2 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 18 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 3-methylpyrazole 2.5 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere.
  • 2-bromo-4-methoxy pyridine 6.7 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 18 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 4-methylpyrazole 2.5 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere.
  • 2-bromo-4-methyl pyridine 6.2 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 18 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 4-methylpyrazole 2.5 g (30 mmol) and potassium tertiary butoxide 4.0 g (36 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere.
  • 2-bromo-4-methoxy pyridine 6.7 g (36 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 18 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • the reaction mixture was filtered under reduced pressure to remove remaining solvents and it was washed with ethanol and distilled water to obtain a white solid.
  • the white solid and ethylene glycol 20 mL were added in a 50 mL one-neck flask, and it was heated to 190 °C and stirred for 30 minutes.
  • the reaction mixture was cooled to a room temperature and the ethylene glycol solvent was removed through distillation under reduced pressure to finally obtain 2-(5-R-2 H- 1,2,4-triazol-3-yl)pyridine of a yellow solid. (0.22 g, 9%)
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and ammonium hydroxyl chloride 7.0 g (0.1 mol) and potassium hydroxide 6.0 g (0.1 mol) were added in methanol 100 ml, and it was heated to 100 °C and stirred for 30 minutes.
  • the produced potassium chloride was concentrated under reduced pressure and removed, and pyridine carbonitrile 7.0 g (60 mmol) was added to the filtered reaction solution and heated to 100 °C, and stirred for 1 hour. After completing the reaction, the mixture was concentrated under reduced pressure and washed with distilled water to obtain hydroxy picolinimidamide of a transparent solid. (9.0 g, 65%)
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and hydroxy picolinimidamide 1.0 g (7.3 mmol), pyridine 1.0 g (12.3 mmol), and acetyl chloride 0.7 g (8.8 mmol) were added to tetrahydrofuran 60 ml, and it was heated to 110 °C and stirred for 8 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3). Organic layers were concentrated under reduced pressure and after removing solvents, 5-methyl-3-(pyridin-2-yl)-1 ,2,4-oxadiazole of a transparent solid was obtained. (0.85 g, 72%)
  • the filtrate was dropped in a sodium dithionite 1.0 M aqueous solution (30 mL), thereby obtaining precipitates of the reduced osmium complex.
  • the produced solid was filtered under reduced pressure and washed with water and acetonitrile several times, and then dried in a vacuum oven to obtain a yellow-brown final compound osmium complex. (0.6 g, 70%)
  • the filtrate was dropped in a sodium dithionite 1.0 M aqueous solution (200 mL), thereby obtaining precipitates of the reduced osmium complex.
  • the produced solid was filtered under reduced pressure and washed with water several times, and then dried in a vacuum oven of 40 °C to obtain a final compound osmium complex. (0.4 g, 56%)
  • Tetraethylene glycol monomethyl ether 2.0 g (9.6 mmol) and tetrabromomethane 3.8 g (11.5 mmol) were added in a 250 mL round bottom flask, and dissolved in dichloromethane 50 mL, and then stirred at 0 °C using an ice tank. After that, during maintaining 0 °C, triphenylphosphine 3.0 g (11.5 mmol) was subdivided for 15 minutes and slowly added and stirred at a room temperature for 2 hours. After completing the reaction, the reaction mixture was extracted with water (100 mL) and dichloromethane (100 mL X 3).
  • the filtrate was dropped in a sodium dithionite 1.0 M aqueous solution (10 mL), thereby obtaining precipitates of the reduced osmium complex.
  • the produced solid was filtered under reduced pressure and washed with water several times, and then dried in a vacuum oven of 40 °C to obtain a black-purple final compound osmium complex. (0.1 g, 56%)
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and sodium azide 1.3 g (19.2 mmol) , 2-ethynyl pyridine 2.0 g (19.2 mmol) and copper sulfate 96 mg (0.38 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 40mL in an argon gas atmosphere.
  • argon degassing was performed for 15 minutes, and then it was heated to 140 °C and stirred for 3 hours.
  • a reflux condenser and a gas inlet were equipped to a 100 mL two-neck round bottom flask, and 2-(1 H -tetrazole-5-yl)pyridine 1.0 g (6.8 mmol) prepared in 1) above was added and dissolved in an anhydrous tetrahydrofuran (30mL) in an argon gas atmosphere, and then sodium hydride 0.4 g (10 mmol) was added. This reaction mixture was stirred at a room temperature for 30 minutes, and iodomethane 1.5 g (10 mmol) was added in an argon gas atmosphere, and then it was heated to 80 °C and stirred for 3 hours.
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 1 H -1,2,4 triazole 3.0 g (43 mmol) and potassium tertiary butoxide 5.8 g (52 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 40mL in an argon gas atmosphere. 2-fluoropyridine 5.0 g (52 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 4 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • the filtrate was dropped in a sodium dithionite 1.0 M aqueous solution (30 mL), thereby obtaining precipitates of the reduced osmium complex.
  • the produced solid was filtered under reduced pressure and washed with water and acetonitrile several times, and then dried in a vacuum oven to obtain a crimson final compound osmium complex. (0.3 g, 62%)
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 1 H -1,2,3 triazole 3.0 g (43 mmol) and potassium tertiary butoxide 5.8 g (52 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 40mL in an argon gas atmosphere. 2-fluoropyridine 5.0 g (52 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 4 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • the filtrate was dropped in a sodium dithionite 1.0 M aqueous solution (30 mL), thereby obtaining precipitates of the reduced osmium complex.
  • the produced solid was filtered under reduced pressure and washed with water and acetonitrile several times, and then dried in a vacuum oven to obtain a green final compound osmium complex. (0.4 g, 69%)
  • the filtrate was dropped in a sodium dithionite 1.0 M aqueous solution (30 mL), thereby obtaining precipitates of the reduced osmium complex.
  • the produced solid was filtered under reduced pressure and washed with water and acetonitrile several times, and then dried in a vacuum oven to obtain a green final compound osmium complex. (0.2 g, 25%).
  • a reflux condenser and a gas inlet were equipped to a 250 mL two-neck round bottom flask, and 3,4-dimethylpyrazole 2.1 g (22 mmol) and potassium tertiary butoxide 2.5 g (22 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 20mL in an argon gas atmosphere.
  • 2-bromo-4-methyl pyridine 3.5 g (20 mmol) was added to this reaction mixture and heated to 100 °C in an argon gas atmosphere and stirred for 18 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (100 mL) and ethylacetate (100 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 50 mL two-neck round bottom flask, and 3,4-dimethylpyrazole 0.6 g (6 mmol) and potassium tertiary butoxide 0.7 g (6 mmol) were added and dissolved in an anhydrous dimethylsulfoxide 8mL in an argon gas atmosphere.
  • 2-bromo-4-methoxy pyridine 1.0 g (5. mmol) was added to this reaction mixture and heated to 80 °C in an argon gas atmosphere and stirred for 6 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and extracted with water (50 mL) and ethylacetate (50 mL X 3).
  • a reflux condenser and a gas inlet were equipped to a 50 mL two-neck round bottom flask, and 4-dimethylamino-2-bromo pyridine 1.0 g (5.0 mmol), 4-methylpyrazole 1.2 g (15 mmol), cooper iodide 0.14 g (0.75 mmol), L-proline 0.17 g (1.5 mmol) and cesium carbonate 4.1 g (12.5 mmol) were added and dissolved in an anhydrous dimethylformamide 20 mL in an argon gas atmosphere. This reaction mixture was heated to 120 °C and stirred for 20 hours.
  • Example 2 Synthesis of oxidation-reduction polymer comprising the transition metal complex according to the present invention
  • Example 3 Synthesis of oxidation-reduction polymer comprising the transition metal complex according to the present invention and a crosslinkable functional group
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 28] polymer prepared in Example 2.1. of 0.2 g was added and completely dissolved in methanol in an argon gas atmosphere.
  • 2-bromoethylamine 20 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 30] polymer prepared in Example 2.3. of 0.2 g was added and completely dissolved in methanol in an argon gas atmosphere.
  • 2-bromoethylamine 20 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 31] polymer prepared in Example 2.4. of 0.4 g was added and completely dissolved in methanol in an argon gas atmosphere.
  • 2-bromoethylamine 50 mg (0.25 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 34] polymer prepared in Example 2.7. of 0.4 g was added and completely dissolved in methanol in an argon gas atmosphere.
  • 2-bromoethylamine 50 mg (0.25 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 35] polymer prepared in Example 2.8. of 0.2 g was added and completely dissolved in methanol in an argon gas atmosphere.
  • 2-bromoethylamine 20 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 40] polymer prepared in Example 2.13. of 0.2 g was added and completely dissolved in methanol in an argon gas atmosphere. 2-bromoethylamine 20 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 41] polymer prepared in Example 2.14. of 0.2 g was added and completely dissolved in methanol in an argon gas atmosphere. 2-bromoethylamine 20 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 42] polymer prepared in Example 2.15. of 0.2 g was added and completely dissolved in methanol in an argon gas atmosphere. 2-bromoethylamine 20 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 43] polymer prepared in Example 2.16. of 0.4 g was added and completely dissolved in methanol in an argon gas atmosphere. 2-bromoethylamine 30 mg (0.15 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 34] polymer prepared in Example 2.7. of 0.4 g was added and completely dissolved in methanol in an argon gas atmosphere.
  • Diethylene glycol-2-bromoethylmethylether 24 mg (0.1 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • a reflux condenser, a gas inlet and a thermometer were equipped to a 100 mL three-neck round bottom flask, and the [Chemical formula 44] polymer prepared in Example 2.17. of 0.4 g was added and completely dissolved in methanol in an argon gas atmosphere. 2-bromoethylamine 30 mg (0.15 mmol) was added to this reaction mixture and heated to 80 °C and stirred for 24 hours. After completing the reaction, the reaction mixture was cooled to a room temperature and dropped in diethyl ether to obtain polymer precipitates. The produced solids were filtered under reduced pressure and washed with diethyl ether several times.
  • the transition metal complex according to the present invention had various potential values depending on the ligand type.
  • an electrochemical sensor electrochemical biosensor
  • an electron transfer mediator of the oxidation-reduction polymer comprising an electron transfer mediator of the oxidation-reduction polymer according to the present invention
  • the potential ( E 0 ) of the electrode to which the oxidation-reduction polymer according to the present invention was applied had a lower potential than the comparison group electrode. This is the result similar to Experimental example 1, and in addition, through this experiment, it was confirmed that the working voltage of the electrochemical sensor for continuous blood glucose measurement could be stably used even at a lower voltage than the comparison group.

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